Abnormal liver carbohydrate and fat metabolism contribute to poor glucose and lipid homeostasis in a variety of metabolic diseases. For this reason, factors that regulate these metabolic pathways in the liver have been intensely studied, yet remain incompletely understood. Commonly, the expression of gluconeogenic enzymes, in particular phosphoenolpyruvate carboxykinase (PEPCK), are thought to control the rate of gluconeogenesis; however, how flux through these pathways change in response to enzyme expression (i.e. control strength) remains poorly understood. Our work demonstrates that in mice with graded levels of PEPCK expression, PEPCK control strength is weak, implying that other factors coordinate control of gluconeogenesis. One of these factors is the rate of hepatic energy production via fat oxidation. For instance, exposure of liver to high levels of fatty acids results in increased gluconeogenesis, and more recently, molecular factors have been identified that coordinate the enzymes of gluconeogenesis and fat oxidation in parallel. We've found that the rate of hepatic TCA cycle flux, a pathway intimately linked to hepatic energy production, correlates more strongly with flux through PEPCK than PEPCK enzyme expression itself. To continue our studies of these pathways we will measure metabolic fluxes in liver in response to altered expression of the gluconeogenic enzymes pyruvate carboxylase (PC) and PEPCK to determine their capacity to influence the rate of gluconeogenesis. Finally, since elevated fat delivery to liver is known to increase gluconeogenesis, and presumably flux through PC and PEPCK, we will also measure hepatic fluxes in response to altered fat availability. These studies will be performed using a multidisciplinary approach comprised of gene altered models, isolated organ preparations and rodent micro-surgery. Nuclear magnetic resonance (NMR) isotopomer analysis will be used to measure metabolic fluxes and these techniques will be corroborated by simultaneous hepatic mass balance determinations. Aberrant fluxes through metabolic pathways of the liver participate in the morbidity of numerous metabolic diseases. Work funded by this grant will substantially enhance our understanding of how metabolic pathways in the liver, specifically gluconeogenesis and fat oxidation, respond to changes in enzyme or substrate concentration. Ultimately, this knowledge is critical for development and interpretation of molecular or pharmacological interventions that modulate these pathways, either by design or happenstance.